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Current Trends in Biotechnology and PharmacyVol. 5 (2) 1193-1205 April 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)Calcium Addition Potentially Reverses Lead and ManganeseInduced Enzymatic and Behavioral Alterations in RatsM. Ram Kumar, K. Praveen Kumar, V. Kavitha and G. Rajarami Reddy*Department of Zoology, Sri Venkateswara University, Tirupati – 517502, India*For Correspondence - gottipolu2002@yahoo.com1193AbstractChronic exposure to lead (Pb) ormanganese (Mn) is known to alter variety ofneurological and behavioral functions. In thisstudy, we have examined the neuro-behavioralperturbations in rats exposed to both Pb and Mn,and the protective effect of calcium supplement.Rats (3 months old) were exposed to Pb (0.2%through drinking water) and Mn (intraperitoniallydaily at a concentration of 2.5 mg/kg body wt)for a period of 3 weeks.A separate batch of animals receivedcalcium (0.02%) in drinking water together withPb and exposed to Mn. Following exposures,the neurochemical alterations were assessed bydetermining the activity changes in synaptosomalacetyl cholinesterase (AChE) and mitochondrialATPases in different brain regions (cortex,hippocampus and cerebellum). Behavioral studiesincluded both cognitive (water maze tasks) andnon-cognitive (open field, exploratory andlocomotory tasks). Combined exposure to Pb andMn resulted in behavioral dysfunctions (decreasein latency, swim distance, swim speed in watermaze task and decrease in open field behaviorand locomotor activity). Similar to the changesobserved in motor and cognitive behavior,decrease in the activity of both AChE andATPases were also observed in the brain regionsof Pb+Mn exposed rats. However, the animalswhich received calcium together with Pb+Mnshowed reversal effect in behavioral as well asthe activity of the enzymes suggesting protectiverole of calcium supplementation against the Pband Mn induced neurotoxicity. The results of thisstudy support our earlier findings on the protectiverole of calcium and zinc against Pb and Mninduced neurotoxicity.Keywords: Behavioral perturbations, Watermaze, Lead, Manganese, Locomotor activity,cholinergic system, Calcium protection.IntroductionThe nervous system is the primary targetfor the low levels of Pb-exposure and thedeveloping brain appears to be especiallyvulnerable to Pb/Mn-neurotoxicity (1-6). Pbexposureat very low doses produces seriousadverse effects on the central nervous system ofchildren and infants, and these effects last forseveral years (7). Perinatal exposure to low levelsof Pb has been shown to exert behavioral andneurochemical alterations in both suckling andadult rats (8). Pb is known to exert its neurotoxiceffects by competing with calcium for calciumreceptors coupled with second messengerfunctions (9) and in some cases, to inhibit theactions of Ca 2+ as a regulator of cell function (9).Calcium protection against lead and manganese toxicity
Current Trends in Biotechnology and PharmacyVol. 5 (2) 1193-1205 April 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1194Manganese (Mn) is an essential tracemetal that is present in all tissues of mammals.This element can act both as a nutrient and atoxicant to the brain (10). The higher intake ofMn, either through the air or the diet, may resultin severe pathologies, particularly in the centralnervous system (CNS) leading to manganism, aParkinson-like disorder (11). The toxic effects ofMn have been widely studied using animalmodels, notably concerning the accumulation ofMn in tissues and interactions with various otherelements (e.g., iron [Fe], zinc [Zn] copper [Cu])after subchronic or chronic administration (12).The adverse effects of Mn on thenervous system probably result from the failureof protective enzymes capable to detoxify criticalamounts of Mn or to alter its oxidation potential.A multifactor hypothesis is more likely, and couldinvolve iron-induced oxidative stress and thedirect interaction of Mn with dopaminergicterminal mitochondria, leading to selectivemitochondrial dysfunction and subsequentexcitotoxicity (13,14). In this study, we haveexamined the alterations in brain enzymes andbehavioral perturbations in rats exposed to bothlead and manganese since both metals coexist inenvironment originating from different exposuresources. We have also studied the protectiveeffects of calcium against the alterations causedby Pb+Mn.Materials and MethodsProcurement and maintenance ofexperimental animals: Young albino rats(Wistar) of 3 months age were purchased fromIISc, Bangalore and maintained in the animalhouse of Dept. of Zoology, S.V. University. Theanimals were housed in clear plastic cages withhardwood bedding in a room maintained at 28 o ±2 o C and relative humidity 60 ± 10% with a 12hour light/day cycle. The animals were fed withstandard pellet diet supplied by Sri VenkateswaraTraders, Bangalore and water ad libitum.Animal exposure to Pb and Mn: Animals wereexposed to 0.2% lead acetate (Sigma) by addingPb- acetate to de-ionized drinking water and Mn(2.5 mg/kg) (Sigma) intraperitonially daily for aperiod of 21 days. Control rats received onlydeionized water without Pb, intraperitonially saline(0.9%) injections daily up to 21 days.Calcium (Ca 2+ ) was supplemented as0.02% in 0.2% Pb- water. The behavioral tasksin control, Pb+Mn-exposed and calciumsupplemented 3 months old rats were observedin the open-field, water maze, exploratorybehavior chamber and locomotory activityrecorder. The brain regions (cerebral cortex,cerebellum and hippocampus) of control, Pb+Mnexposed,and Ca 2+ supplemented rats werecollected and used for biochemical assays.Behavioural StudiesOpen-Field Behavior: The open field test hasbeen widely used to assess emotional reactivity/anxiety. It provides measures of locomotoractivity. The horizontally directed activity (orlocomotion) is measured by the number of linecrossings, and vertically directed activity (orexploration) is measured by the frequency ofrearings (15). The open-field behaviour of threemonths age rats was assessed in a wooden boxmeasuring 90 x 90 x 30 cm high. The floor of thearena was divided into 36 equal squares by blacklines. Immediately after a rat was placed in thecentre of the open field, the movement of theanimal was scored. The number of squarescrossed with all paws (crossings), the standingson the hind legs (rearings), placing the nose againstwall or floor (sniffing), wiping, licking, combingor scratching of any part of the body (grooming)were counted in all sessions. All the activitiesmeasured were combined together to assess themean total behaviour in each session. Testing wascarried out on five consecutive days in five minutesessions in control, Pb-exposed and supplementedanimals.Ram Kumar et al
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